Yersinia pestis is the causative agent of plague which has caused widespread loss of human life during recurrent pandemics. On average approximately 2500 cases of human plague occur world-wide each year. Recently over 98% of the plague cases are found in Africa. In nature, the bacterium cycles between mammals (mainly rodents) and fleas. The interaction of plague with fleas is facilitated by the formation of a biofilm. In the oriental rat flea, Xenopsylla cheopis, formation of the biofilm leads to blockage of the valve between the esophagus and stomach of the flea, known as the proventriculus. Blockage of the proventriculus may extend the transmission time. The hemin-storage genes (hms) of Y. pestis are responsible for the production of a biofilm which is essential for flea blockage. The Hms+ phenotype was initially characterized as the ability of cells grown at 26:C but not 37:C, to bind exogenous hemin or the dye, Congo red, and is due to the production of an exopolysaccharide matrix or biofilm. There are at least 5 genes involved in the synthesis of the Hms- dependent biofilm; hmsHFRS, hmsT, and hmsP. Two of these gene products, HmsP and HmsT, are responsible for the synthesis and turnover of the signaling molecule, cyclic di-guanylic acid. Over-production of HmsT, a diguanylate cyclase, or the absence of HmsP, a phosphodiesterase, leads to increased biofilm formation. Polyamines also play a role in the creation of a Y. pestis biofilm. Mutants that are unable to synthesize polyamines can not form a biofilm unless supplied with exogenous putrescine. This proposal will continue studies on the mechanisms that control biofilm development and the protein-protein interactions needed for biofilm formation. The biochemical functions of the Hms proteins as well as the role of the biofilm in fleas and mice will be addressed. In specific aim 1, the interaction between Y. pestis wild-type cells as well as various hms mutants and different flea species will be investigated. The role of c-di-GMP in virulence of plague will also be examined. Specific factors which regulate biofilm development will be studied in specific aim 2. In specific aim 3, the interactions between the Hms proteins as well as their biochemical and enzymatic characteristics will be addressed. The proposed studies will provide new insights into the regulation of plague biofilm formation and its role in the transmission of the disease from fleas to mammals. Systems similar to that responsible for plague biofilm are present in a number of diverse bacteria. Biofilms are often associated with the ability to survive harsh conditions as well as increased resistance to antibiotics. Understanding the mechanisms involved in producing the plague biofilm as well as the factors that regulate its production will likely be relevant for biofilm formation in other pathogens.